Alpha 2HS glycoprotein for treatment of cancer and a method for preparation thereof

ABSTRACT

This invention characterizes the selective apoptotic activity of specially prepared Alpha2 and fragments thereof. Specifically, Alpha2 which has been overloaded with zinc, as well as fragments thereof, selectively induce apoptosis in HT-29 (colon cancer), LNCaP (prostate cancer), and Hep G2 (Hepatoma) cells, while having no affect on CCD 18 Co (normal colon) cells.

CLAIM OF PRIORITY

[0001] This application is a continuation-in-part to U.S. Ser. No.10/145,682 (filed May 14, 2002) which is a continuation-in-part to U.S.Ser. No. 09/902,208 (filed Jul. 09, 2001), which is acontinuation-in-part to U.S. Ser. No. 09/414,136 (filed Oct. 07, 1999),now U.S. Pat. No. 6,258,779 (issued Jul. 10, 2001), which is acontinuation-in-part to U.S. Ser. No. 09/149,878 (filed Sep. 08, 1998),now U.S. Pat. No. 5,994,298 (issued Nov. 30, 1999), which is acontinuation-in-part to U.S. Ser. No. 08/993,432 (filed Dec. 18, 1997).

BACKGROUND OF THE INVENTION

[0002] Human beings have had a long battle against cancer. Because thedisease is so widespread, manifests itself in so many different ways andis so relentless, the potential market for effective cancer therapies isenormous. It is estimated that 10 million people in the U.S. either haveor have had cancer. The National Cancer Institute (NCI) projected thatin 1995, some 1.2 million new cases of cancer will be diagnosed in theUnited States, and that 538,000 people will die of the disease.

[0003] Cancer is currently treated, with a low degree of success, withcombinations of surgery, chemotherapy and radiation. The reason for thelow degree of success in chemotherapy is because currentchemotherapeutic approaches target rapidly dividing tumor cells. Thisapproach is ineffective against cancer that is dormant or slow growing.Such treatments also affect other, noncancerous cells that dividerapidly, thereby causing harmful side effects.

[0004] Recently, a new approach has emerged in the battle againstcancer. This approach is based on the biological phenomenon called“Apoptosis”. Apoptosis is also called “programmed cell death” or “cellsuicide”. (Krammer, et al., “Apoptosis in the APO-1 System”, Apoptosis:The molecular Basis of Cell Death, pp. 87-99 Cold Spring HarborLaboratory Press, 1991). In contrast to the cell death caused by cellinjury, apoptosis is an active process of gene-directed, cellularself-destruction that serves a biologically meaningful function. (Kerr,J. F. R and J. Searle J. Pathol. 107:41, 1971). One example of thebiologically meaningful function of apoptosis occurs during themorphogenesis of an embryo. (Michaelson, J. Biol. Rev. 62:115, 1987).Just as the sculpting of a sculpture needs the addition as well asremoval of clay, the organ formation (Morphogenesis) of an embryo relieson cell growth (addition of clay) as well as cell death (removal ofclay). As a matter of fact, apoptosis plays a key role in the human bodyfrom the early stages of embryonic development through to the inevitabledecline associated with old age. (Wyllie, A. H. Int. Rev. Cytol. 68:251,1980). The normal functioning of the immune, gastrointestinal, andhematopoietic systems depend upon the normal function of apoptosis. Whenthe normal function of apoptosis goes awry, the result can be one of anumber of diseases including cancer, viral infections, auto-immunedisease/allergies, neurodegeneration, or cardiovascular diseases.Because of the role apoptosis plays in human diseases, apoptosis isbecoming a prominent buzzword in the pharmaceutical research field. Hugeamounts of time and money are being spent in an attempt to understandhow it works, how it can be encouraged or inhibited, and what this meansfor practical medicine. A handful of companies have been formed with theprime direction of turning work in this nascent field into marketablepharmaceutical products. The emergence of a core of innovative youngcompanies combined with the steps being taken by established industrialplayers are certain to make apoptosis research one of thefastest-growing and most promising areas of medical study.

[0005] The idea that cancer may be caused by insufficient apoptosisfirst arose in the early 1990's (Cope, F. O. and Wille, J. J.,“Apoptosis”: The Molecular Basis of Cell Death, Cold Spring HarborLaboratory Press, p. 61, 1991). This idea opened a door for a newconcept in cancer therapy—Cancer cells may be killed by encouragingapoptosis. Apoptosis modulation, based on the processes present innormal development, is a potential mechanism for controlling the growthof tumor cells. Inducing apoptosis in tumor cells is an attractiveapproach because, at least in theory, it would teach the cells to commitsuicide. Nevertheless, since the objective of cancer treatment is tokill cancer cells without killing the host, the success of thistreatment is still dependent on the availability of drugs that canselectively induce apoptosis in tumor cells without affecting normalcells. In this patent application, the apoptotic activity of naturallyoccurring fetuin (which has been modified), chemically synthesizedfetuin, recombinant fetuin, and their associated fragments upon cancercells are elucidated. In addition, the effects of alpha 2 and associatedfragments are expounded. These proteins and polypeptides may present anew class of anticancer drugs that induce apoptosis in cancer cells,which may offer a breakthrough in cancer therapy.

SUMMARY OF THE INVENTION

[0006] The purpose of this invention is to demonstrate the selectiveapoptotic activity of fetuin, alpha 2 and associated fragments.Specifically, recombinant fetuin from E. Coli was found to induceapoptotic activity in cancer cells at concentrations of 1 μM. Inaddition, the active fragments from recombinant fetuin induced apoptosisin cancer cells at a concentration of 2.5 μM. Modified Alpha 2-HSglycoprotein induced apoptosis at LD₅₀ (dosage for the induction of 50%cell death) at 6 hours on HT-29, Hep G2, and LNCaP at 0.75 μM, 0.87 μM,and 1.5 μM respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 shows a table of test data of using fetal fetuin on micebearing leukemia.

[0008]FIG. 2 shows a table of the comparison of original fetuin withzinc in comparison with the supercharged zinc fetuin to reach LD₅₀(dosage for the induction of 50% cell death).

[0009]FIG. 3 shows another table of the comparison of original fetuinwith zinc in comparison with the supercharged zinc fetuin to reach LD₅₀.

[0010]FIG. 4 shows a slide of LNCaP (prostate cancer) cells withouttreatment of filtrate containing SEQ ID NO: 1.

[0011]FIG. 5 shows a slide of LNCaP cells incubated with filtratecontaining SEQ ID NO: 1 for six (6) hours.

[0012]FIG. 6 shows an additional slide of LNCaP (prostate cancer) cellswithout treatment of filtrate containing SEQ ID NO: 1.

[0013]FIG. 7 shows a slide of LNCaP cells incubated with filtratecontaining SEQ ID NO: 1 and which expressed membrane blebbing, which isan indicator of cells undergoing apoptosis.

[0014]FIG. 8 shows a table illustrating, among other things, the effectof incubating the filtrate containing SEQ ID NO: 1 treated withproteinase K.

[0015]FIG. 9 shows a slide of LNCaP cells without filtrate containingSEQ ID NO: 1.

[0016]FIG. 10 shows a slide of LNCaP cells, which were incubated withfiltrate containing SEQ ID NO: 1 for three (3) hours and expressedmembrane “blebbing,” which is an indicator of cells undergoingapoptosis.

[0017]FIG. 11 is a graph of SEQ ID NO: 1 (nM) versus the percentapoptosis in HT-29, CCD-18 Co, and LNCaP cells.

[0018]FIG. 12 is a graph of the time of incubation of SEQ ID NO: 1filtrate versus percent apoptosis in HT-29 and CCD-18 Co cells.

[0019]FIG. 13 is a table of the LD₅₀ values for fetuin and variousfetuin fragments, including SEQ ID NO: 1.

[0020]FIG. 14 shows a slide of HT-29 cells incubated with 5 μM Alpha2-HS Glycoprotein for 4 hours (stained by Hoeschst dye).

[0021]FIG. 15 shows a slide of HT-29 cells without Alpha 2-HSGlycoprotein (stained by Hoeschst dye).

[0022]FIG. 16 shows another slide of HT-29 cells incubated with 5 μMAlpha 2-HS Glycoprotein for 4 hours (stained by Hoeschst dye).

[0023]FIG. 17 shows another slide of HT-29 cells without Alhpha2.

[0024]FIG. 18 shows a slide of LNCaP cells incubated with 5 μM Alpha2-HS Glycoprotein for 4 hours.

[0025]FIG. 19 shows a slide of LNCaP cells without Alpha 2-HSGlycoprotein.

[0026]FIG. 20 shows a slide of Hep G2 cells incubated with 5 μM Alpha2-HS Glycoprotein for 4 hours.

[0027]FIG. 21 shows a slide of Hep G2 cells without Alpha 2-HSGlycoprotein.

[0028]FIG. 22 shows a slide of CCD 18 Co cells incubated with 5 μM Alpha2-HS Glycoprotein for 6 hours.

[0029]FIG. 23 shows a slide of CCD 18 Co cells without Alpha 2-HSGlycoprotein.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Initially, five proteins were isolated: Apogen P-1a, Apogen 1b,Apogen 1c, Apogen P-2 and Apogen L.

(A) Isolation of Apogen P-1

[0031] (1) Source of Apogen P-1

[0032] Apogen P-1 was isolated from the conditioned medium of a cellline called XC, which was derived from rat tumor (ATCC CCL 165). XCcells were first grown in Dulbecco's Modification of Eagle's Medium(DMEM) containing 10% Fetal bovine serum (FBS) for 3 days. XC cells werethen washed with PBS (3×100 ml) to remove serum and then grown in DMEMcontaining no FBS for 4 days. From this serum free conditioned medium,an activity inducing apoptosis in a prostate cancer cell line calledLNCAP was detected. On the other hand, normal human lung fibroblast cellline (CCD 39 Lu) and breast cancer cells (MCF-7) were not affected bythis activity.

[0033] (2) Activity of Apogen P-1

[0034] (a) Apoptosis Inducing activity

[0035] The activity of the crude conditioned medium of XC cells wastested on the following cell lines: JEG-3 (Choriocarcinoma), G401(Wilm's tumor) LNCAP (Prostate cancer), T84 (colon cancer), HL-60(leukemia), breast cancer cells (MCF-7), and CCD 39 Lu (normal lungfibroblast). When 10 folds concentrated conditioned medium was incubatedfor 18 hours with the above cell lines in the presence of 5% serum, theconditioned medium induced apoptosis in JEG-3 cells (35%), G 401 cell(27%), LNCaP(100%) and without activity in CCD 39 Lu (0%), T84(0%),MCF-7(0%) and HL-60(0%).

[0036] Apoptosis is a distinct type of cell death that differsfundamentally from degenerative death or necrosis in its nature andbiological significance. A cell undergoing apoptosis is distinct from acell undergoing necrosis both morphologically and biochemically.Morphologically, the earliest definitive changes in apoptosis that havebeen detected with the electron microscope are compaction of the nuclearchromatin into sharply circumscribed, uniformly dense masses about thenuclear envelope and condensation of the cytoplasms. Phase-contrastmicroscopy of cells under apoptosis shows the condensation and thefragmentation of DNA and the blebbing of the cell.

[0037] To morphologically demonstrate that the XC conditioned mediumcontains activity inducing apoptosis, LNCAP cells were incubated withcontrol medium or the conditioned medium treated as described as abovefor 15 hr and then stained with Hoechst dye for 2 hours. The nuclei ofthe LNCAP cells that had been incubated with the control medium werenormal and healthy. However, the nuclei of the LNCAP cells that had beenincubated with the conditioned medium (X20, exchanged to RPMI) showedthe characteristics of apoptosis. First, the conditioned medium causedthe condensation of the nucleus, demonstrated by the more intensefluorescent light compared with the control nucleus. Second, the nucleuscondensation was accompanied by the fragmentation of DNA, asdemonstrated by the breakage of the nucleus. As mentioned above, thecondensation of the nucleus and the DNA fragmentation are themorphological characteristics of cells under apoptosis. These resultssuggest that the conditioned medium from XC cells contain an activityinducing apoptosis in LNCAP cells. On the other hand, the conditionedmedium fails to induce apoptosis in normal human lung fibroblast (CCD 39Lu cells) and breast cancer cells (MCF-7). The nuclei of CCD 39 Lu cellsremain the same with or without incubating with the conditioned mediumof XC cells.

[0038] (b) Cell repelling activity

[0039] The partially purified Apogen P-1b (Q2 anionic exchangerchromatography step) isolated as described below was recently found tocontain an activity other than inducing apoptosis. It was found thatApogen P-1b has an activity that repels cells away. This activity isopposite to that of growth factors; many growth factors such as PlateletDerived Growth Factor (PDGF), Epidermal Growth factor (EGF), FibroblastGrowth factor (FGF) or Transforming Growth factor (TGF) function as a“chemoattractant”—which means that these growth factors attract cellstoward them. (Grotendorst, G. R. et al., Proc. Natl. Acad. Sci. 78:3669,1981; Grant, M. B. et al Invest. Ophthal. Visual Science. 33:3292,1992). This finding suggests that Apogen P-1b isolated in this inventionplays an opposite biological function as that of a growth factor. Thatis, growth factors induce cell growth and attract cells, whereas ApogenP-1b induces cell death and repels cells. Apogen P-1b is the first“chemorepellent” found in the field of modern biology.

[0040] A tissue culture device called Transwell Insert purchased fromCostar (Cambridge, Mass.) was used to discover the chemorepellentactivity of Apogen P-1b. This device, which has been widely used for thestudies of cell migration/invasion, contains an upper chamber and alower chamber. Between these two chambers is a polyester microporousmembrane with 3.0 μm pore size, which allows cells to migrate throughthe membrane. Tested cells are grown on the upper chamber, and thetested compound is placed in the lower chamber. If this tested compoundis a chemoattractant, more cells should migrate through the membranethan the control sample. In our experiments, Hep G2 (100,000 cells)cells, which have a cell size 3-4 times as big as the membrane poresize, were grown in the upper chamber for 2 hours, and then, thepartially purified Apogen-1b (30 μl) isolated by ammonium sulfateprecipitation and Q2 HPLC chromatography as described above was placedin the lower chamber. After 15 hours, cells that had migrated throughthe membrane were collected by treating the membrane with 0.2 ml oftrypsin solution for 30 min. Cells in ten microliters of the trypsinsolution were counted in a hemacytometer. In several experiments, it wasfound that the partially purified Apogen-1b contained an activitydecreasing the number of cells going through the membrane. For example,in one experiment, in the presence of the partially purified ApogenP-1b, the cell number in 10 microliters trypsin solution (which are thecells go through membrane) is 24±4, whereas the number of cells that gothrough the membrane in the control experiment is 82±27. This resultsuggests that the partially purified Apogen P-1b prevents Hep G2 cellsfrom migrating through membrane. To unequivocally show that Apogen P-1brepel cells, an inverted experiment was installed. Instead of placingApogen P-1b in the lower chamber, Apogen P-1b was placed in the upperchamber. After 12 hours, 56±19 cells went through the membrane comparedwith the control experiment of 30±1.7 cells per 10 microliters oftrypsin solution. The statistically significant increase or decrease inthe number of cells going through the membrane by alternatively placingApogen P-1b in the upper or lower chamber of this tissue culture devicestrongly suggests that Apogen P-1b repels cells.

[0041] (3) Isolation of Apogen P-1 from XC conditioned medium

[0042] The Apogen P-1 present in the conditioned medium was isolated bythe following steps:

[0043] Step 1: Ammonium Sulfate Precipitation

[0044] Apogen P-1 was precipitated by 80% saturated of ammonium sulfateby adding 561 g of ammonium sulfate per liter of conditioned medium.Pellet was collected by centrifugation and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4). After removal of ammonium sulfate bydialysis, the dissolved proteins were separated by a Q2 HPLC column.

[0045] Step 2: Q2 HPLC Chromatography

[0046] The dissolved proteins isolated by ammonium sulfate precipitationwere concentrated and loaded on to a Q2 column (Bio-Rad) which wasfurther developed by a linear gradient constructed by buffer A (10 mMTris-HCl, pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl)using BioRad's BioLogic HPLC system. The linear gradient was constructedby increasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% buffer B for 5 min.

[0047] The Apogen P-1 activity was assayed by the induction of apoptosisin LNCAP cells. We found that there were three activity peaks across thechromatogram profile. Fraction 5 to 7 caused 70% cell death; fraction8-10 caused 65% cell death; and fraction 11-14 caused 90% cell death in18 hr. We collected fractions 5-7 and named it Apogen P-1a; fractions8-10 was named Apogen P-1b; and fractions 11 to 14 was named ApogenP-1c. These three Apogen P-1's were further purified by a reverse phasecolumn.

[0048] Step 3: Reverse phase chromatography.

[0049] Apogen P-1a, Apogen P-1b and Apogen P-1c were separatelyconcentrated to 1.5 ml. One ml of methanol containing 0.05%trifluoracetic acid was added. In each sample, a large amount of proteinwas precipitated by this treatment. Whereas, the apoptosis inducingactivity remained in the supernatant. The supernatant was then appliedto a reverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H2O, 0.05% TFA) and solutionB (Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min.

[0050] Step 4: Preparative electrophoresis.

[0051] Apogen 1c isolated by anion exchange chromatography was purifiedby both Reverse phase chromatography (step 3) and PreparativeElectrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad). In thereverse phase chromatogram of Apogen P-1a, fractions 12-13 had activityinducing 80% cell death in LNCAP cells at 10 hr. In the reverse phasechromatogram of Apogen P-1b, fractions 14 and 15 had activity inducing45% cell death in LNCAP cells at 18 hr.

[0052] In the reverse phase chromatogram of Apogen P-1c, fraction No. 5had activity inducing 52% cell death in LNCAP cells at 18 hr.

[0053] The purity of the isolated Apogen P-1a, Apogen P-1b and ApogenP-1c was checked with SDS-polyacrylamide gel electrophoresis stainedwith silver staining.

[0054] (1) Apogen P-1a: a protein band with molecular weight of 70 KDwas obtained. This result suggests the successful purification of ApogenP-1a, which has the molecular weight of 70 KD on SDS-PAGE.

[0055] (2) Apogen P-1b: A single faint protein band with molecularweight of 55 KD was obtained. This result suggests the successfulpurification of Apogen P-1b, which has the molecular weight of 55 KD onSDS-PAGE.

[0056] (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phasechromatography leads to the isolation of a 70 KD protein whereas thepurification of Apogen-1c by preparative electrophoresis leads to thepurification of a 57 KD protein. A major protein band with molecularweight of 70 KD was obtained by Reverse Phase chromatography. A 57 KDprotein, on the other hand, was isolated by preparative electrophoresis.

[0057] The next obvious step would be to obtain enough of the proteinband to sequence it.

(B) Isolation of Apogen P-2

[0058] (1) Source of Apogen P-2

[0059] Apogen P-2 was isolated from the conditioned medium of a cellline called C3H 10T1/2, which was derived from mouse embryo cells (ATCCCCL 226). C3H 10T1/2 cells were first grown in alpha Modification ofEagle's Medium (alpha-MEM) containing 10% Fetal bovine serum (FBS) for 3days. Cells were then washed with PBS (3×100 ml) to remove serum andthen grown in alpha-MEM containing no FBS for 4 days. From this serumfree conditioned medium, we detected an activity inducing apoptosis in aprostate cancer cell line called LNCAP. On the other hand, normal humanlung fibroblast cell line (CCD 39 Lu) was not affected by this activity.

[0060] (2) Activity of Apogen P-2

[0061] (a) Apoptosis Inducing Activity

[0062] The activity of the crude conditioned medium of C3H 10T1/2 cellswas tested on the following cell lines: LNCAP (Prostate cancer), breastcancer cells (MCF-7), and CCD 39 Lu (normal lung fibroblast). When the10-fold concentrated conditioned medium was incubated for 18 hours withthe above cell lines in the presence of 5% serum, the conditioned mediuminduced apoptosis in LNCaP (100%) and without activity in CCD 39 Lu(0%). To morphologically demonstrate that the C3H 10T1/2 conditionedmedium contains activity inducing apoptosis, LNCAP cells were incubatedwith control medium or with the conditioned medium treated as describedas above for 15 hr and then stained with Hoechst dye for 2 hours. Thenuclei of the LNCAP cells that had been incubated with control mediumwere normal and healthy. However, the nuclei of the LNCAP cells that hadbeen incubated with the conditioned medium showed the characteristic ofapoptosis. First, the conditioned medium caused the condensation of thenucleus as demonstrated by the more intense fluorescent light comparedwith the control nucleus. Second, the condensation of the nucleus wasaccompanied by the fragmentation of DNA as demonstrated by breakage ofthe nucleus. As mentioned above, the condensation of the nucleus and DNAfragmentation are the morphological characteristics of cells underapoptosis. The same holds true of breast cancer cells (MCF-7) in which85% apoptotic effect was observed after 18 hours of exposure to P-2.These results suggest that the conditioned medium from C3H10T1/2 cellscontains an activity inducing apoptosis in LNCAP and MCF-7 cells. On theother hand, the conditioned medium failed to induce apoptosis in normalhuman lung fibroblast (CCD 39 Lu cells). The nuclei of CCD 39 Lu cellsremained the same with or without incubating with the conditioned mediumof C3H10T1/2 cells.

[0063] (b) Cell Repelling Activity

[0064] The partially purified Apogen P-2, isolated by ammonium sulfateprecipitation, hydroxylapatite, and heparin treatment as describedabove, was recently found to contain an activity other than inducingapoptosis. Similar to Apogen P-1b, Apogen P-2 has activity that repelscells away. Transwell Insert purchased from Costar (Cambridge, Mass.)was used to discover the chemorepellent activity of Apogen P-2. Thisdevice, which has been widely used for the study of cellmigration/invasion, contains an upper chamber and a lower chamber.Between these two chambers is a polyester microporous membrane with 3.0μm pore size, which allows the cells to migrate through the membrane.The tested cells (HL-60) were grown on the upper chamber, and the testedcompound (Apogen P-2) was placed in the lower chamber. In ourexperiments, HL-60 (100,000 cells) cells, which have a cell size 2-3times as big as the membrane pore size, were grown in the upper chamberfor 2 hours, and then, the partially purified Apogen P-2 (30 μl)isolated by ammonium sulfate precipitation, hydroxylapatite, and Heparinagarose as described above was placed in the lower chamber. After 6hours, cells that had migrated through the membrane were collected fromthe lower chamber; the medium in lower chamber (0.6 ml) was centrifugedfor 10 min; and the HL-60 cells that went through the membrane werecollected and resuspended in 80 μl of PBS. The cells in ten microlitersof the PBS solution were counted in a hemacytometer. In severalexperiments, we found that the partially purified Apogen P-2 containedan activity that decreased the number of cells going through themembrane. For example, in one experiment, in the presence of thepartially purified Apogen P-2, the number of cells in 10 microliters PBSsolution (which are the cells that pass through the membrane) was47±5.6, whereas the number of cells that went through the membrane inthe control experiment was 213±40. At this moment, no apoptosis wasobserved in HL-60 cells present in the upper chamber. This resultsuggests that the partially purified Apogen P-2 prevents the HL-60 cellsfrom migrating through membrane.

[0065] (3) Isolation of Apogen P-2 from C3H10T1/2 Conditioned Medium

[0066] The Apogen P-2 present in the conditioned medium was isolated bythe following steps:

[0067] Step 1: Ammonium sulfate precipitation.

[0068] Apogen P-2 was precipitated by ammonium sulfate (80% saturated)by adding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

[0069] Step 2: Hydroxylapatite treatment.

[0070] After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl(pH 7.5), the dissolved proteins were incubated with Hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

[0071] Step 3: Heparin agarose treatment.

[0072] The supernatant from step 2 was further incubated with Heparinagarose (Sigma) for 1 Hr. After removing HTP gel by centrifugation, theactivity inducing apoptosis in LNCAP cells was found to be present inthe supernatant.

[0073] Step 4: Reverse phase chromatography.

[0074] Apogen P-2 present in the supernatant of Heparin agarose in step3 was further purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%Trifluoracetic acid was added. A large amount of protein wasprecipitated by this treatment. Whereas, the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H2O, 0.05% TFA) and solutionB (Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A in 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min.

[0075] In the reverse phase chromatogram of Apogen P-2, fractions 12-14have activity inducing 80% cell death in LNCAP cells at 12 hr. Thepurity of the isolated Apogen P-2 was checked with SDS-polyacrylamidegel electrophoresis stained with silver staining, and a single proteinband with molecular weight of 65 Kd was obtained.

(C) Isolation of Apogen L

[0076] (1) Source of Apogen L

[0077] Apogen L was isolated from the conditioned medium of XC cell line(ATCC CCL 165). XC cells were grown in Dulbecco's Modification ofEagle's Medium (DMEM) containing 10% Fetal bovine serum (FBS) for 4days. From this conditioned medium, we detected an activity inducingapoptosis in a leukemia cell line called HL-60. On the other hand,normal human lung fibroblast cell line (CCD 39 Lu) was not affected bythis activity.

[0078] (2) Isolation of Apogen L from XC conditioned medium

[0079] The Apogen L present in the conditioned medium was isolated bythe following steps:

[0080] Step 1: DE52 Absorption

[0081] The conditioned medium was incubated with the anion exchanger, DE52 (Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubationmixture was centrifuged and DE 52, which binds Apogen L was collectedand washed with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen Lwas then eluted from DE 52 cellulose by 10 mM Tris-HCl (pH 7.5)containing 0.5 M NaCl.

[0082] Step 2: Heparin Agarose Absorption

[0083] Apogen L isolated as described in step 1 was further absorbed byHeparin agarose (Sigma) by incubating Apogen L with Heparin agarose for1 hr. Heparin agarose was collected by centrifugation and was washedwith 10 mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose wasthen eluted by 2 M NaCl.

[0084] Step 3: Q2 HPLC Chromatography

[0085] Apogen L isolated as described above was concentrated and loadedonto a Q2 column (Bio Rad) which is further developed by a lineargradient constructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B(10 mM Tris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLCsystem. The linear gradient was constructed by increasing buffer B from0% to 100% in buffer A within 10 min. The purity of the isolated ApogenL was checked with SDS-polyacrylamide gel electrophoresis stained withsilver staining. A single protein band with molecular weight of 55 Kdwas obtained.

[0086] (3) Activity of Apogen L

[0087] The activity of Apogen L isolated as described above was testedon the following cell lines: HL-60 (leukemia) and CCD 39 Lu (normal lungfibroblast). To morphologically demonstrate that Apogen L containsactivity inducing apoptosis, HL-60 cells were incubated with Apogen Lisolated as described above for 15 hr and then stained with Hoechst dyefor 2 hours. The nuclei of the HL-60 cells that had been incubated withcontrol medium were normal and healthy. However, the nuclei of the HL-60cells that had been incubated with Apogen L showed the characteristic ofapoptosis. First, Apogen L caused the condensation of nucleus asdemonstrated by the more intense fluorescent light compared with thecontrol nucleus. Second, the nucleus condensation was accompanied by thefragmentation of DNA as demonstrated by the breakage of nucleus. Asmentioned above, nucleus condensation and DNA fragmentation are the twomorphological characteristics of cells under apoptosis. These resultssuggest that the isolated Apogen L contains an activity inducingapoptosis in HL-60 cells. Apogen L also induces apoptosis in MCF-7(breast cancer) cells. On the other hand, the conditioned medium failsto induce apoptosis in normal human lung fibroblast (CCD 39 Lu cells).

EXAMPLES A. Methods

[0088] 1. Preparation of Condition Media.

[0089] A. Preparation of XC Condition Medium for Isolation of ApogenP-1.

[0090] Apogen P-1 was isolated from the conditioned medium of a cellline called XC, which was derived from a rat tumor (ATCC CCL 165). XCcells were first seeded in a roller bottle (Polystyrene, areasurface=850 Cm2, Corning) in Dulbecco's Modification of Eagle's Medium(DMEM) containing CO2, 10% fetal bovine serum (FBS), nonessential aminoacids, penicillin and streptomycin for 3 days. XC cells were then washedwith PBS (3×100 ml) to remove serum and then grown in 100 ml of DMEMcontaining no FBS (with CO2), nonessential amino acids, penicillin andstreptomycin) for 4 days. The conditioned medium was collected andclarified by centrifugation.

[0091] B. Preparation of C3H 10T1/2 Condition Medium for Isolation ofApogen P-2.

[0092] Apogen P-2 was isolated from the conditioned medium of a cellline called C3H10T1/2, which was derived from a mouse embryo and waspurchased from American Type Culture Collection (ATCC CCL 226).C3H10T1/2 cells were first seeded in a roller bottle (Polystyrene, areasurface=850 Cm2, Corning) in alpha Modification of Eagle's Medium(alpha-MEM) containing CO2, 10% Fetal bovine serum (FBS), penicillin andstreptomycin for 3 days. C3H10T1/2 cells were then washed with PBS(3×100 ml) to remove serum and then grown in 100 ml of alpha MEMcontaining no FBS (with CO2, penicillin and streptomycin) for 4 days.The conditioned medium was collected and clarified by centrifugation.

[0093] C. Preparation of XC condition medium for isolation of Apogen L.

[0094] Apogen L was isolated from the conditioned medium of a cell linecalled XC, which was derived from rat tumor (ATCC CCL 165). XC cellswere first seeded in a roller bottle (Polystyrene, area surface=850 Cm2,Corning) in Dulbecco's Modification of Eagle's Medium (DMEM) containingpenicillin, streptomycin, CO2, nonessential amino acids and 10% Fetalbovine serum (FBS) for 4 days. The conditioned medium was collected andclarified by centrifugation.

[0095] 2. Assays

[0096] (a) Cell Death (Apoptosis) Assay

[0097] Prostate cancer cell line LNCAP was routinely used for theisolation of Apogen P-1 and Apogen P-2, whereas leukemia cell line HL-60was used for the isolation of Apogen L. The methods of assays were asfollows: LNCAP or HL-60 (1,000 cells) was seeded in 10 microliters RPMIcontaining 15% or 20% Fetal bovine serum, penicillin and streptomycin at37 degrees, 5% CO2 in Microtray plates (25 μl wells, Robbins ScientificCorp.). The tested sample (10 μl) was added 3-4 hours after cells wereseeded. After incubation of the tested sample with cells for 15 hours,two microliters of Hoechst dye (0.03 ng/ml in PBS) was added. Two hourslater, cells that were stained with Hoechst dye were examined underfluorescence microscope. The nuclei of apoptotic cells showed DNAcondensation and fragmentation, which were easily identified by Hoechstdye staining. The percentage of apoptotic cells was calculated by thefollowing equation:${\% \quad {Apoptotic}\quad {cells}} = \frac{\begin{matrix}{{Number}\quad {of}\quad {cells}\quad {with}\quad {DNA}} \\{{condensation}\quad {and}\quad {fragmentation}}\end{matrix}}{{Total}\quad {cell}\quad {number}}$

[0098] (b) Cell Repelling Assay

[0099] There are two reasons that Hep G2 cells were chosen for the studyof cell repelling activity. First, Hep G2 cells are not sensitive toApogen P-1 in inducing apoptosis. Second, the cell size of Hep G2 cellis about 3-4 times as big as the pore size of the membrane on theTranswell Insert, which is a good cell size for cell migration/invasionstudy. A tissue culture device called Transwell Insert purchased fromCostar (Cambridge, Mass.) was used to discover the chemorepellentactivity of Apogen P-1b. This device, which has been widely used for thestudies of cell migration/invasion, contains an upper chamber and alower chamber. Between these two chambers is a polyester microporousmembrane with 3.0 μm pore size, which allows cells to migrate throughthe membrane. Tested cells were grown on the upper chamber, and thetested compound was placed in the lower chamber. If this tested compoundis a chemoattractant, more cells will migrate through membrane than thecontrol sample. In our experiments, Hep G2 (100,000 cells) cells, whichhave a cell size 3-4 times as big as the membrane pore size were grownin the upper chamber (Minimum Essential Medium Eagle containing 10% FBS,PS and nonessential amino acid, 0.1 ml) for 2 hours, and then thepartially purified Apogen-1b (30 μl) isolated by ammonium sulfateprecipitation and Q2 HPLC chromatography as described above was placedin the lower chamber which contained 0.6 ml of the same growth mediumfor Hep G2 cells. After 15 hours, cells that had migrated through themembrane were collected by treating the membrane with 0.2 ml of trypsinsolution for 30 min. Cells in ten microliters of the trypsin solutionwere counted in a hemacytometer.

[0100] 3. Protein Isolation

[0101] A. Isolation of Apogen P-1

[0102] Step 1: Ammonium Sulfate Precipitation

[0103] Apogen P-1 was precipitated by 80% saturated of ammonium sulfateby adding 561 g of ammonium sulfate per liter of XC conditioned medium.The pellet was collected by centrifugation, and the proteins weredissolved in 10 mM Tris-HCl (pH 7.4). After removal of ammonium sulfateby dialysis, the dissolved proteins were separated by a Q2 HPLC column.

[0104] Step 2: Q2 HPLC Chromatography

[0105] The dissolved proteins isolated by ammonium sulfate precipitationwere concentrated and loaded onto a Q2 column (Bio Rad) which wasfurther developed by a linear gradient constructed by buffer A (10 mMTris-HCl, pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl)using BioRad's BioLogic HPLC system. The linear gradient was constructedby increasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% buffer B for 5 min. The Apogen P-1 activity was assayed by theinduction of apoptosis in LNCAP cells. Three activity peaks were foundacross the chromatogram profile. Fractions 5 to 7 caused 70% cell death;fractions 8-10 caused 65% cell death; and fractions 11-14 caused 90%cell death in 18 hr. We collected fractions 5-7 and named it ApogenP-1a; fractions 8-10 was named Apogen P-1b; and fractions 11-14 wasnamed Apogen P-1c. These three Apogen P-1's were further purified by areverse phase column.

[0106] Step 3: Reverse Phase Chromatography.

[0107] Apogen P-1a, Apogen P-1b and Apogen P-1c were separatelyconcentrated to 1.5 ml. One ml of methanol containing 0.05%Trifluoracetic acid was added. In each sample, a large amount of proteinwas precipitated by this treatment. Whereas, the apoptosis inducingactivity remained in the supernatant. The supernatant was then appliedto a reverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H2O, 0.05% TFA) and solutionB Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min. (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min.

[0108] Step 4: Preparative Electrophoresis

[0109] Apogen 1c isolated by anion exchange chromatography was purifiedby both Reverse Phase Chromatography (step 3) and PreparativeElectrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad). In thereverse phase chromatogram of Apogen P-1a, fractions 12-13 had activityinducing 80% cell death in LNCAP cells at 10 hr.

[0110] In the reverse phase chromatogram of Apogen P-1b, fractions 14and 15 had activity inducing 45% cell death in LNCAP cells at 18 hr.

[0111] In the reverse phase chromatogram of Apogen P-1c, fraction No. 5had activity inducing 52% cell death in LNCAP cells at 18 hr.

[0112] The purity of the isolated Apogen P-1a, Apogen P-1b and ApogenP-1c were checked with SDS-polyacrylamide gel electrophoresis stainedwith silver staining.

[0113] (1) Apogen P-1a: A protein band with molecular weight of 70 KDwas obtained. This result suggests the successful purification of ApogenP-1a, which has a molecular weight of 70 KD on SDS-PAGE.

[0114] (2) Apogen P-1b: A single faint protein band with molecularweight of 55 KD was obtained. This result suggests the successfulpurification of Apogen P-1b, which has a molecular weight of 55 KD onSDS-PAGE.

[0115] (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phasechromatography lead to the Isolation of a 70 KD protein, whereas thepurification of Apogen 1c by preparative electrophoresis leads to thepurification of a 57 KD protein. A major protein band with molecularweight of 70 KD was obtained by Reverse Phase chromatography. A 57 KDprotein, on the other hand, was isolated by preparative electrophoresis.

[0116] B. Isolation of Apogen P-2

[0117] The Apogen P-2 present in C3H10T1/2 conditioned medium wasisolated by the following steps:

[0118] Step 1: Ammonium sulfate precipitation.

[0119] Apogen P-2 was precipitated by ammonium sulfate (80% saturated)by adding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

[0120] Step 2: Hydroxylapatite treatment.

[0121] After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl(pH 7.5), the dissolved proteins were incubated with hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

[0122] Step 3: Heparin agarose treatment.

[0123] The supernatant from step 2 was further incubated with Heparinagarose (Sigma) for 1 hr. After removing HTP gel by centrifugation, theactivity inducing apoptosis in LNCAP cells was found to be present inthe supernatant.

[0124] Step 4: Reverse phase chromatography.

[0125] Apogen P-2 present in the supernatant of Heparin agarose in step3 was further purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%trifluoacetic acid was added. Large amounts of proteins wereprecipitated by this treatment. Whereas, the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc.) and developed by alinear gradient constructed by solution A (H2O, 0.05% TFA) and solutionB Methanol (0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min. In the reverse phase chromatogram of ApogenP-2, fractions 12-14 have activity inducing 80% cell death in LNCAPcells at 12 hr. The purity of the isolated Apogen P-2 was checked withSDS-polyacrylamide gel electrophoresis stained with silver staining. Asingle protein band with molecular weight of 65 Kd was obtained.

[0126] C. Isolation of Apogen L

[0127] The Apogen L present in the conditioned medium was isolated bythe following steps:

[0128] Step 1: DE52 Absorption

[0129] The conditioned medium was incubated with the anion exchanger, DE52 (Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubationmixture was centrifuged, and DE 52, which binds Apogen L, was collectedand washed with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen Lwas then eluted from DE 52 cellulose by 10 mM Tris-HCl (pH 7.5)containing 0.5 M NaCl.

[0130] Step 2: Heparin Agarose Absorption

[0131] Apogen L isolated as described in step 1 was further absorbed byHeparin agarose (Sigma) by incubating Apogen L with Heparin agarose for1 hr. Heparin agarose was collected by centrifugation and was washedwith 10 mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose wasthen eluted by 2 M NaCl.

[0132] Step 3: Q2 HPLC Chromatography

[0133] Apogen L isolated as described above was concentrated and loadedonto a Q2 column (Bio Rad) which was further developed by a lineargradient constructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B(10 mM 22 Tris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLCsystem. The linear gradient was constructed by increasing buffer B from0% to 100% in buffer A in 10 min. The purity of the isolated Apogen Lwas checked with SDS-polyacrylamide gel electrophoresis stained withsilver staining. A single protein band having activity with a molecularweight of approximately 55 Kd was obtained.

[0134] 4. Isolation of Bovine Fetuin as a Component of Protein P-2 andthe Apoptotic Effect Thereof in Tumor Cell Lines.

[0135] The observation that Apogen P-1a, P-1b, P-1c, P-2 and L wereisolated from embryonic cell lines led us to speculate that newborn orembryonic tissue may secrete “Apogen,” which may selectively induceapoptosis in tumor cell lines. Based on this speculation, our attentionturned towards a protein named “Fetuin” for the following reasons: (1)Fetuin is mainly a fetal protein, in the sense that the highestconcentrations are found in serum and body fluids of embryos andfetuses. For example, the concentration of fetuin in bovine serumdrastically decreases, probably within a few days after birth, to 1-2%of the fetal level. (Yang, et al., Biochim. Biophy. Acta. 1130, 149-1561992). (2) A histochemical study has shown that fetuin may controltissue remodelling and physiological cell death during embryonicdevelopment. (Von Bulow, et al., Histochemistry 99:13-22, 1993). Thesefeatures of fetuin suggest the possibility that fetuin may containactivity inducing cell death (apoptosis).

[0136] Additionally, a protein with an amino acid sequence identical toFetuin was isolated from the preparation of Apogen P-2. Thus, thecomposition of Apogen P-2 consists at least in part of fetuin. Thereforefetuin was prepared and tested for apoptotic activity. Interestingly, itwas found that only bovine fetuin prepared by a special method was ableto induce apoptosis in tumor cell lines. The commercial fetuin that isprepared by ammonium sulfate precipitation and EDTA treatment was foundto contain a very low activity in inducing apoptosis in tumor cells.

[0137] 4A. Preparation of Bovine Fetuin.

[0138] Bovine fetuin was prepared by the modified Spiro method (Spiro R.G., Journal of Biological Chemistry 235, 10: 2860, 1960) according tothe following steps:

[0139] 1. One hundred milliliters of Fetal Bovine Serum (FBS).

[0140] 2. Add two hundred milliliters of 0.05 M Zinc Acetate containing30% (V/V) ethanol, adjust to pH 6.4 by 1M NH4OH—NH4Cl, let stand 15hours at −5° C.

[0141] 3. Collect the supernatant by centrifugation, add 1.0 M BariumAcetate and 95% ethanol to give 0.03 M Barium Acetate, 25% ethanol. Letstand 2 hours at −5° C.

[0142] 4. Collect the supernatant by centrifugation, add 95% ethanol togive 40% ethanol. Let stand 15 hours at −10° C.

[0143] 5. Collect the precipitate. Dissolve the pellet by phosphatebuffer saline.

[0144] The purified fetuin showed a single protein band with apparentmolecular weight of 63 Kd on SDS-PAGE.

[0145] 4B. Induction of Apoptosis in Tumor Cell Lines Using BovineFetuin.

[0146] Fetuin purified from fetal bovine serum by the proceduredescribed above was dissolved in phosphate buffer saline (PBS). The freeZinc Acetate and Barium Acetate were removed by repetitiveconcentration. Fetuin was tested in LNCaP and HL-60 cells. LNCaP orHL-60 (1,000 cells) was seeded 10 microliters RPMI containing 15% or 20%Fetal bovine serum, penicillin and streptomycin at 37 degree, 5% CO2 inmicrotray plates (25 μl wells, Robbins Scientific Corp.). Fetuin (in 10μl PBS) at concentration of 100 ng/ml was added 3-4 hours after thecells were seeded. After incubation of the tested sample with the cellsfor 15 hours, two microliters of Hoechst dye (0.03 ng/ml in PBS) wasadded. Two hours later, cells that were stained with Hoechst dye wereexamined under fluorescence microscope. The nuclei of apoptotic cellsshowed DNA condensation and fragmentation, which were easily identifiedby Hoechst dye staining. The percentage of apoptotic cells wascalculated by the following equation:${\% \quad {Apoptotic}\quad {cells}} = \frac{\begin{matrix}{{Number}\quad {of}\quad {cells}\quad {with}\quad {DNA}} \\{{condensation}\quad {and}\quad {fragmentation}}\end{matrix}}{{Total}\quad {cell}\quad {number}}$

[0147] The nuclei of the LNCaP cells that were incubated with controlsample (PBS) were normal and healthy. However, the nuclei of the LNCaPcells that were incubated with fetuin (100 ng/ml in PBS) showed thecharacteristics of apoptosis. First, the cells in the presence of fetuinshowed condensation of the nucleus as demonstrated by the more intensefluorescent light compared with the control nucleus. Second, the nucleuscondensation was accompanied by the fragmentation of DNA as demonstratedby breakage of the nucleus. As condensation of the nucleus and DNAfragmentation are the two morphological characteristics of cells underapoptosis, these results suggest that fetuin contains an activityinducing apoptosis in LNCaP cells. Furthermore, the nuclei of the HL-60cells that were incubated with control buffer (PBS) were normal andhealthy. However, the nuclei of the HL-60 cells that were incubated withfetuin showed the characteristics of apoptosis. Fetuin causedcondensation of the nucleus as demonstrated by the more intensefluorescent light compared with the control nucleus. Second, the nucleuscondensation was accompanied by the fragmentation of DNA as demonstratedby breakage of the nucleus. As mentioned above, the nucleus condensationand DNA fragmentation are the two morphological characteristics of cellsunder apoptosis. Accordingly, these results suggest that fetuin containsan activity inducing apoptosis in HL-60 cells.

[0148] 4C. Bovine Fetuin Selectively Induces Apoptosis in Cancer CellsWithout Having an Effect on Normal Cell Lines.

[0149] The effect of fetuin on the induction of apoptosis was comparedin various cell lines. At a concentration of 50 μg/ml, fetuin preparedas described above strongly induced apoptosis in tumor cell lines suchas: LNCaP (prostate cancer), PC-3 (prostate cancer), HL-60 (leukemia),MCF-7 (breast cancer), Colo 205 (colon cancer), Calu-1 (lung cancer).Normal lung fibroblast (CCD 39 Lu), on the other hand, is not affectedby fetuin.

[0150] Fetuin at 25 μg/ml highly induced apoptosis in LNCaP, HL-60cells, and MCF-7 cells while it was found to be inactive in inducingapoptosis in CCD 39 Lu cells. Fetuin (25 μg/ml) prepared as describedabove was incubated with CCD 39 Lu cells grown in MEM in a microtrayplate for 15 hours. The CCD 39 Lu cells remained morphologicallyunchanged in the presence of fetuin.

[0151] 4D. Only Fetuin Prepared by the Method Described Above is Able toInduce Apoptosis in Tumor Cell Lines.

[0152] Fetuin purchased from Sigma has a very low activity in inducingapoptosis in LNCaP cells. For the fetuin purchased from Sigma, apoptosisinducing activity was observed only at a very high concentration (>250μg/ml) and at long incubation time (2 days). However, fetuin (25 μg/ml)prepared by the method described in Section 4A above induced apoptosisin LNCaP cells by up to 90% in 4 hours. It was initially estimated thatthe apoptotic activity of fetuin prepared as described in Section 4Aabove is more than fifty thousand folds higher than that of fetuinprepared by other methods.

[0153] In the years of research following the original findings, it hasbeen determined that fetuin from Sigma induces apoptosis at a very highconcentration and at a long incubation time. It is conservativelyestimated that the fetuin as prepared in Section 4A has more than onehundred times greater apoptotic activity than fetuin prepared by othermethods. While this is not as dramatic as the 50,000 times increase asoriginally reported, it still represents a significant apoptoticadvantage over previously available fetuin in terms of incubation timeand LD₅₀ values (dosage for the induction of 50% cell death).

[0154] Sigma's fetuin is prepared by methods which include ammoniumsulfate precipitation and EDTA treatment. Both of these treatments maycause deprivation of the Zinc ion from the protein, which may cause theirreversible loss of the protein's apoptotic activity.

[0155] 4E. Effect of Fetal Fetuin on Leukemia Cells In Vivo.

[0156] The previous data demonstrate that fetuin induces apoptosis incancer cells in vitro. The data provided below shows that the in vivotesting of fetuin in mice having leukemia was successful. The resultsshow that fetuin has an anti-leukemia effect in mice. FIG. 1 shows theincrease in survival of leukemia-bearing mice treated with fetal fetuin.

[0157] Method

[0158] Forty DBA/2 female mice (17-20 grams; Simonsen Laboratories,Inc., Gilroy, Calif.) kept on a standard diet and water ad libitum wereinoculated with tumor cell line P388D1 (ATCC cell line number CCL46).The mice were randomly segregated into groups of ten (10). Zinc-chargedfetal fetuin (10 mg/ml) were intraperitoneally injected into group I at0.002 ml/mouse, group II at 0.02 ml/mouse and group III at 0.2 ml/mouse.Group IV was the control group, which was injected with 0.5 ml of salinesolution. The injections were continued for 10 days. Mortalities wererecorded for 60 days. The results were expressed as the percentageincrease in life span (ILS): ${ILS} = \frac{\begin{matrix}{{100 \times {Median}\quad {Life}\quad {Span}\quad {Treated}} -} \\{{Median}\quad {Life}\quad {Span}\quad {Controlled}}\end{matrix}}{{Life}\quad {Span}\quad {Controlled}}$

[0159]FIG. 1 shows that while 100% of the untreated leukemia-bearingmice were dead after 24 days, 80% of the mice treated with a high doseof fetuin, namely 100 mg/Kg of fetal fetuin, survived more than 58 days.This in vivo experiment demonstrates that mice bearing leukemia that aretreated with fetal fetuin have an increased life span of 141%.

[0160] 4F. Method of Preparing Supercharged Zinc Fetuin:

[0161] The method of preparing fetuin with zinc was refined andimproved. As stated above, fetuin prepared by the method as described inSection 4A above is able to induce apoptosis in tumor cell lines.However, commercial fetuin such as that from Sigma is found to have avery low activity in inducing apoptosis in tumor cells and in inducingapoptosis in LNCaP cells. For fetuin from Sigma, apoptosis inducingactivity was observed only at a very high concentration (>250 μg/ml) andat a long incubation time (2 days), whereas fetuin (25 μg/ml) asprepared in Section 4A above induced apoptosis in LNCaP cells by up to90% in 4 hours. It was initially estimated that the apoptotic activityof fetuin as prepared as described in Section 4A is more than fiftythousand times higher than that fetuin prepared by other methods. Theradically different results suggest a fundamental difference in thechemical composition of commercially available fetuin and the fetuinprepared in accordance with the procedure in Section 4A.

[0162] In the years of research following the original findings, it wasobserved that fetuin from Sigma induced apoptosis at a very highconcentration (>>5 mM) and at a long incubation time (2 days), whereasfetuin (approximately 50 μM) as prepared in Section 4A above inducedapoptosis in LNCaP cells by up to 90% in 4 hours. It is conservativelyestimated that the fetuin as prepared in Section 4A has more than onehundred times greater apoptotic activity than fetuin prepared by othermethods. While this is not as dramatic as the 50,000 times increase asoriginally reported, it still represents a significant apoptoticadvantage over previously available fetuin in terms of incubation timeand LD₅₀ values (dosage for the induction of 50% cell death).

[0163] After looking at the methods of preparing commercially availablefetuin, it was found that ammonium sulfate precipitation and EDTAtreatment was used in preparing fetuin. It was speculated that thisammonium sulfate precipitation and EDTA treatment might causedeprivation of the ions from the protein causing irreversible loss ofthe protein's apoptotic activity. However, it was not known whether itwas the loss of zinc alone, or in combination with the loss of anotherion(s), that caused the decreased apoptotic activity in commerciallyavailable fetuin. While it was determined that Fetuin-Ca is inactive ininducing apoptosis (data not shown), and barium occurs in only traceamounts, to determine which ion, or combination of ions, were mosteffective in increasing the apoptotic ability of fetuin, fetuin asprepared in Section 4A above was treated with a chelating agent, such asEDTA, to strip all the inorganic ions, including zinc, calcium, andbarium from the protein. After removing these inorganic ions, the“naked” fetuin was treated or incubated with 0.5 M Zinc Acetate toreload or to bind the fetuin with zinc only. The results of thisrefinement process are shown in FIGS. 2-3. LD₅₀ (dosage for theinduction of 50% cell death) concentrations on LNCaP cells incubated for6 hours reveal that the improved preparation of Fetuin-Zn or“supercharged zinc fetuin” enhances fetuin's ability to induce apoptosisin cancer cells by three to four times as compared with the originalfetuin bound with zinc as prepared in Part 4A. It is hypothesized thatthe fetuin previously bound up with calcium and barium created aninactive form of the protein. By stripping out all ions and replacingthem with zinc, inactive fetuin molecules were converted to active form,thereby explaining the dramatic increase in apoptotic activity. Suchsupercharged zinc fetuin is a valuable step forward in the fight againstcancer.

[0164] In one preferred embodiment of this preparation process:

[0165] 1. Incubation Mixture: 700 μg of fetuin (0.2 ml; as prepared bythe method as described above in Section 4A) was incubated with 0.5 mlof 50 mM EDTA for approximately one (1) hour.

[0166] 2. Concentration: Add 1.5 ml of saline solution to thisincubation mixture and concentrate to near dryness using a molecularsieve and centrifugal force. Repeat this procedure four (4) times, sothat most of the inorganic ions and EDTA are removed. This “naked”fetuin will be retained on the top of the filter (molecular sieve).

[0167] 3. Incubate the “naked” fetuin (0.2 ml) with 0.5 ml of 0.5 M ZincAcetate for approximately three (3) hours.

[0168] 4. Remove the free Zinc Acetate using the combination of thesaline solution, the molecular sieve, and centrifugal force as describedin Step 2 above.

[0169] 5. A Specific Peptide Fragment From Fetuin-Zinc That CausesApoptosis In Cancer Cells.

[0170] a. Preparation of Fetuin Fragment:

[0171] As described above in Section 4F (Method of PreparingSupercharged Zinc Fetuin), supercharged zinc fetuin was prepared bypre-treatment of fetal bovine fetuin with a chelating agent (EDTA) toremove the inorganic ions, including zinc, calcium, and barium ions,from the fetuin. The resulting stripped fetuin was incubated with 0.5 MZinc Acetate in order to “supercharge” or load the fetuin with zinc.

[0172] Three hundred (300) micrograms of supercharged zinc fetuin wasdissolved in a 50 μl saline solution and then dried in a tube under avacuum. It is hypothesized that this drying step breaks apart thesupercharged zinc fetuin into peptide fragments.

[0173] The dried fragments (of supercharged zinc fetuin) werereconstituted in 50 μl water. This fragment solution was passed througha molecular sieve membrane having a molecular weight cut-off of 10,000daltons. The resulting filtrate of fragments was collected and tested oncells in an apoptosis assay. As shown in FIG. 5, when LNCaP cells areincubated with the supercharged zinc fetuin fragment filtrate for six(6) hours, the LNCaP cells detach and die. Compared with the control(LNCaP with no filtrate) as shown in FIG. 4, FIG. 5 shows thatincubation of the prostate cancer cells with the supercharged zincfetuin fragment filtrate causes apoptosis of the cancer cells. FIG. 7shows that the supercharged zinc fetuin fragment filtrate treated LNCaPcells also exhibit membrane “blebbing,” which is a characteristictypical of cells undergoing apoptosis. FIG. 6, which shows LNCaP cellswithout the supercharged zinc fetuin fragment filtrate, lacks thismembrane “blebbing” and characteristic of apoptosis.

[0174] b. Is Protease Sensitive.

[0175] Additionally, the apoptosis-inducing activity of the superchargedzinc fetuin fragment filtrate was found to be protease sensitive.Incubation of the supercharged zinc fetuin fragment filtrate withproteinase K completely removed the apoptosis-inducing activity.Proteinase K is an enzyme that cleaves peptide bonds.

[0176] After preparing “supercharged” zinc fetuin as stated in Section4F above, the resulting composition was dried in a tube and under avacuum. This dried supercharged zinc fetuin was reconstituted in 50 μlof water. This solution was filtered through a molecular sieve membrane(Centricon 10 tube with a molecular weight cut-off: 10,000 daltons). Thefiltrate was collected and treated with 5 μl (1 unit/μl) proteinase Kfor three (3) hours at 37° C. After treatment with proteinase K, thetreated filtrate was filtered through a molecular sieve membrane(Centricon 10 tube) in order to remove the proteinase K. The proteinaseK was retained by the membrane, and the treated filtrate passed throughthe membrane.

[0177] To test the effect of a protease on the apoptotic activity of thefiltrate, the filtrate treated with proteinase K was tested on cancercells. These results were compared to the filtrate that was not treatedwith proteinase K.

[0178] The effect of proteinase K on the apoptotic ability of theFetuin-Zinc fragments is summarized in FIG. 8. Experiments 1 and 2 wereconducted with one set of supercharged zinc fetuin fragment filtrate,and Experiment 3 was conducted with another set of supercharged zincfetuin fragment filtrate. FIG. 8 shows that incubation with a proteaseseems to inactivate the apoptotic effect of the zinc charged fetuinfragment. Because a protease, such as proteinase K, cleaves peptidebonds, the test results of FIG. 8 strongly suggest that a peptide or aprotein of fetuin is responsible for the induction of apoptosis incancer cells.

[0179] c. The Filtrate Contains Two Major Peptides Derived from Fetuin.

[0180] The dried and reconstituted filtrate was found to contain peptidefragments. The amino acid sequence analysis revealed two major peptidefragments in the filtrate:

[0181] (1) H-T-F-S-G-V-A-S-V-E (amino acid no. 300-309; His Thr Phe SerGly Val Ala Ser Val Glu; SEQ ID NO: 1) and

[0182] (2) S-A-S-G-E-A-F-H (amino acid no. 310-317; Ser Ala Ser Gly GluAla Phe His; SEQ ID NO: 2) of fetuin.

[0183] To identify which of these peptide fragments was responsible forthe apoptosis-inducing activity, the two fragments (H-T-F-S-G-V-A-S-V-E,amino acid no. 300-309, SEQ ID NO: 1 and S-A-S-G-E-A-F-H amino acid no.310-317, SEQ ID NO: 2) of the full-length fetuin molecule) werechemically synthesized. Upon in vitro testing of these chemicallysynthesized peptide fragments, SEQ ID NO: 1 was shown to have thegreater apoptotic activity. LNCaP (prostate cancer cells) were incubatedwith SEQ ID NO: 1. In FIG. 10, chemically synthesized SEQ ID NO: 1caused membrane “blebbing” in LNCaP cells after three (3) hours ofincubation. Incubation of SEQ ID NO: 2 with LNCaP cells did not show anyapoptotic activity or membrane “blebbing.” FIG. 9 shows the control ofLNCaP cells without SEQ ID NO: 1. These results suggest that the peptidefragment that induced apoptosis and that was present in the filtratecorresponds to amino acid no. 300-309, SEQ ID NO: 1 of full-lengthfetuin.

[0184] 6. Characterization Of SEQ ID NO: 1.

[0185] a. SEQ ID NO: 1 Selectively Induced Apoptosis In Cancer Cells ButNot In Normal Cells.

[0186] Previously, it was found that fetuin and supercharged zinc fetuininduced apoptosis in various cancer cells without affecting certainnormal cells. To test whether SEQ ID NO: 1 derived from fetuin retainsthis selectivity in inducing apoptosis in cancer cells only while notaffecting normal cells, various concentrations of SEQ ID NO: 1 weretested on HT-29 (colon cancer), CCD-18 Co (normal colon), and LNCaP(prostate cancer) cells. As shown in FIG. 11, SEQ ID NO: 1 inducedapoptosis in HT-29 and LNCaP cells without affecting CCD-18 Co cells.These results suggest that the fragment was similar to fetuin andsupercharged zinc fetuin in selectively inducing apoptosis in cancercells while not affecting normal cells.

[0187] b. SEQ ID NO: 1 Rapidly Induced Apoptosis in HT-29 (Colon Cancer)Cells.

[0188]FIG. 12 shows the effect of time on the induction of apoptosis bySEQ ID NO: 1. At four (4) hours, SEQ ID NO: 1 at a concentration of 0.4μM induced apoptosis in 52% of the HT-29 colon cancer cells. At six (6)hours, almost all of the HT-29 cells were induced to apoptosis. Even theslightest increase in incubation time (from 4 to 6 hours), dramaticallyincreased the apoptotic affect of SEQ ID NO: 1.

[0189] c. The Peptide Fragment Derived From Fetuin Is More Potent ThanFull Length Fetuin or Supercharged Zinc Fetuin in Inducing Apoptosis.

[0190] Previously, the LD₅₀ (dosage for the induction of 50% cell death)at 6 hours for full-length supercharged zinc fetuin in LNCaP cells wasdetermined to be 3-10 μM. Preliminary results for the LD₅₀ at 6 hoursfor the supercharged zinc fetuin filtrate were determined to be 0.3-0.4μM. Therefore, a much smaller amount of fragment was required to induceapoptosis in cancer cells than is required with full-length superchargedzinc fetuin. Further, SEQ ID NO: 1 is more potent in inducing apoptosisthan its parent molecule (FIG. 13).

[0191] Taking into consideration the previous estimates:

[0192] (1) Fetuin as prepared in Section 4A above is approximately 100times more powerful than fetuin prepared from other methods;

[0193] (2) “Supercharged” Zinc Fetuin is approximately three to fourtimes more powerful than the fetuin as prepared in Section 4A (see FIGS.2-3); and

[0194] (3) SEQ ID NO: 1 is approximately eight to ten times morepowerful than “Supercharged” Zinc Fetuin (see FIG. 13),

[0195] it is estimated that SEQ ID NO: 1 is approximately severalthousand times more powerful than fetuin prepared from other methods.

[0196] The most current results reveal a minor variation from theoriginal results depending on whether the peptide sequence is chemicallysynthesized, versus whether it is a fragment derived from superchargedzinc fetuin. SEQ ID NO: 1 derived from supercharged zinc fetuin has anLD₅₀ at 6 hours of approximately 0.7 μM as compared to SEQ ID NO: 1 thatis chemically synthesized which has an LD₅₀ at 6 hours of approximately2.5 μM.

[0197] 7. Other Sequences Of Fetuin From Other Sources.

[0198] In addition, peptide sequences were determined from other animalsera, including pig, sheep and mice. K. M. Dziegielewska, et. al.,Fetuin, 16-17, (R. G. Landes Co. 1995). These peptide sequences have asimilarity of 60-90% with the fetuin isolated from bovine serum. Thesesimilar fetuin peptide sequences also have valuable apoptotic activity.The peptide sequences for SEQ ID NO: 1 for other species are: Human(H-T-F-M-G-V-V-S-L-G; His Thr Phe Met Gly Val Val Ser Leu Gly; SEQ IDNO:3); Pig (H-S-F-S-G-V-A-S-V-E; His Ser Phe Ser Gly Val Ala Ser ValGlu; SEQ ID NO:4); Sheep (H-T-F-S-G-V-A-S-V-E; His Thr Phe Ser Gly ValAla Ser Val Glu; SEQ ID NO:5); Rat (H-T-F-S-G-V-A-S-V-E; His Thr Phe SerGly Val Ala Ser Val Glu; SEQ ID NO:6); and Mouse (H-A-F-S-P-V-A-S-V-E;His Ala Phe Ser Pro Val Ala Ser Val Glu; SEQ ID NO:7). Id.

[0199] The LD₅₀ at 6 hours for the chemically synthesized fetuinfragments for SEQ ID NO's: 3, 4, and 7 are 1.0 μM, 0.3 μM and 0.5 μMrespectively. The LD₅₀ at 6 hours for the chemically synthesized fetuinfragments for SEQ ID NO's: 1, 5 and 6 are 2.5 μM. A summary of theseresults reflect that fetuin as prepared in the methods taught hereinhave selective apoptotic activity. In addition, active fragments fromnaturally occurring fetuin that have been modified as suggested hereinor fragments chemically synthesized also have selective apoptoticactivity.

[0200] 8a. Recombinant Fetuin

[0201] The studies described above demonstrated the apoptotic activityof fetuin and its fragments. It was observed that when the carbohydratemoiety of natural fetuin was removed by certain enzymes, then theapoptotic inducing activity was boosted 2-3 fold. Accordingly,experiments were done to test the apoptotic activity of recombinantfetuin expressed in E. Coli. The E. Coli-expressed fetuin had apoptoticactivity with an LD₅₀ at 6 hours of 1 μM. This is about 5 times greaterthan the apoptotic activity of supercharged zinc fetuin as prepared inSection 4F above.

Example

[0202] 1. Molecular cloning of the bovine fetuin gene.

[0203] Fetal bovine tissue was obtained commercially. mRNA was thenisolated (Frederick, M. A. et al., “Short Protocols in MolecularBiology” pp. 5-12, John Wiley & Sons, 2^(nd) Ed. 1992). Full length cDNAwas reverse transcribed with poly(t) and a plasmid library constructed.

[0204] cDNA clones coding for fetuin were screened. The bovine fetuingene was isolated by traditional methods and subcloned into pBluescriptvector. The gene was confirmed by DNA sequencing.

[0205] The gene contains one open reading frame encoding 359 amino acidresidues, which is homologous with other fetuin genes. The confirmedgene was ligated into vector pCRT7-NT vector and junction was confirmedby both restriction digestion and sequencing (Frederick, M. A. et al.,“Short Protocols in Molecular Biology” p. 3, John Wiley & Sons, 2^(nd)Ed. 1992). The gene was prepared from confirmed clone in E. coli TOP10strain.

[0206] Over 10 different clones were identified. Protein expression wasperformed in 1 L and 5 L culture. Culture was grown at 34 C to OD600about 0.5-0.6 and then 1 mM IPTG to induce expression for 4-5 hours.Precipitate bacteria pellet for protein purification. Bacteria pelletwas suspended into 20 ml of 1X binding buffer and sonicated on ice. Runthe lysate through columns. The eluted protein was dialysed against twochanges of 1X PBS at 4C. Further protein purification was carried byHPLC chromatography.

[0207] b. Recombinant Fragment

[0208] From E. Coli expressed fetuin, peptide fragments were generatedas in Section 5a. Three hundred (300) micrograms of the zinc chargedfetuin was dissolved in a 50 μl saline solution and then dried in a tubeunder a vacuum. The dried fragments were reconstituted in 50 μl water.This fragment solution was passed through a molecular sieve membranehaving a molecular weight cut-off of 10,000 daltons.

[0209] This peptide filtrate strongly induced apoptosis in LNCaP andHT-29 cells without affecting normal colon cells (CCD 19 Co). Thepeptide filtrate caused DNA condensation and DNA fragmentation in HT-29cells. The onset of the induction of cell death by the fragment was veryrapid. In as soon as 60 minutes, cell death was observed. The LD₅₀ at 6hours of this recombinant peptide filtrate is 2.5 μM.

[0210] 9. Supercharged Alpha 2 HS-glycoprotein

[0211] The previous studies demonstrated selective apoptotic activity infull length fetuin (as modified in the methods described above) andfragments (whether from modified natural fetuin, chemically synthesized,or recombinantly generated).

[0212] The human version of fetuin is called Alpha 2HS-glycoprotein(discovered by J. F. Heremans and K. Schmid in 1961). The amino acidsequence of this protein is about 60% homologous to bovine fetuin.Bovine fetuin and human Alpha 2-HS glycoprotein are truly specieshomologues and not simple (closely) related proteins. Alpha 2-HSglycoprotein is a glycoprotein found in human serum. The concentrationin plasma is higher in fetal (145 mg/100 ml) than in adult (40-60 mg/100ml). It is known that this protein binds calcium and barium with highaffinity. Fetuin levels in human plasma are regulated in the manner of anegative acute phase reactant. As a negative acute-phase reactant,reduced levels of human Alpha 2HS-glycoprotein have been observed inseveral diseases including Paget's disease of bone (4), various cancers(5-8), and severe malnutrition and infection (9-12).

[0213] Since fetuin and its fragments as modified above inducedapoptosis, it was hypothesized that the human counterpart of fetuin,Alpha 2-HS glycoprotein, also induced apoptosis. Like fetuin, Alpha 2-HSglycoprotein, modified by the addition of zinc, also selectively inducedapoptosis. Specifically, Alpha 2-HS glycoprotein supercharged with zincwas found to induce apoptotic activity in cancer cells at concentrationsof 0.75 μM. Since the concentration of Alpha 2-HS glycoprotein in adulthuman serum is about 0.5 mg/ml (8.3 μM), the 0.75 μM apoptotic thresholdis a concentration that can easily be reached by IV administration orother means such as oral administration. It is believed thatsupercharged zinc Alpha 2-HS glycoprotein and its active fragments maybe used to treat cancer along the same lines as immunoglobulins are usedfor the treatment of certain humans diseases, by first preparing theproteins from human sera and then re-administering them into humanpatients after active manipulation of the proteins.

[0214] This present section teaches the selective apoptotic activity ofAlpha 2-HS glycoprotein in its naturally occurring (modified bysupercharging with zinc), chemically produced, and/or recombinantlyproduced forms. In addition, this section teaches the fragments of Alpha2-HS glycoprotein also have apoptotic activity whether from the modifiednatural Alpha 2-HS glycoprotein, chemically synthesized, orrecombinantly generated fragments.

[0215] 9a. Purification Of Alpha2-HS Glycoprotein From Adult Serum

[0216] Alpha 2-HS glycoprotein was prepared by a modified Spiro method(Spiro R. G., Journal of Biological Chemistry 235, 10: 2860, 1960)according to the following steps:

[0217] 1. One hundred milliliters of human serum (from donor orcommercial such as CALBIOCHEM)

[0218] 2. Add two hundred milliliters of 0.05 M Zinc Acetate containing30% (V/V) ethanol, adjust to pH 6.4 by 1M NH4OH—NH4Cl, let stand 15hours at −5° C.

[0219] 3. Collect the supernatant by centrifugation, add 1.0 M BariumAcetate and 95% ethanol to give 0.03 M Barium Acetate, 25% ethanol. Letstand 2 hours at −5° C.

[0220] 4. Collect the supernatant by centrifugation, add 95% ethanol togive 40% ethanol. Let stand 15 hours at −10° C.

[0221] 5. Collect the precipitate by centrifugation. Dissolve the pelletby phosphate buffer saline.

[0222] The purified fetuin showed a single protein band with apparentmolecular weight of 63 Kd on SDS-PAGE.

[0223] 9b. Method Of Preparing Supercharged Zinc Alpha 2-HS glycoprotein

[0224] In one preferred embodiment of this preparation process:

[0225] 1. Incubation Mixture: 700 μg of Alpha 2-HS glycoprotein (0.2 ml;as prepared by the method as described above in Section 9A) wasincubated with 0.5 ml of 50 mM EDTA for approximately one (1) hour.

[0226] 2. Concentration: Add 1.5 ml of saline solution to thisincubation mixture and concentrate to near dryness using a molecular ormolecule sieve and centrifugal force. Repeat this procedure four (4)times, so that most of the inorganic ions and EDTA are removed. This“naked” fetuin will be retained on the top of the filter (molecularsieve).

[0227] 3. Incubate the “naked” fetuin (0.2 ml) with 0.5 ml of 0.5 M ZincAcetate for approximately three (3) hours.

[0228] 4. Remove the free Zinc Acetate using the combination of thesaline solution, the molecular sieve, and centrifugal force as describedin Step 2 above.

[0229] 9c. Supercharged Zinc Alpha2-HS Glycoprotein Induced Apoptosis InLNCaP, HT-29 And Hep G2 Without Affecting CCD 18Co

[0230] Supercharged zinc Alpha 2-HS glycoprotein induces apoptosisselectively. The morphological changes in HT-29 cancer cell lines due toapoptosis are shown in FIGS. 14 and 16. Incubation of HT-29 cells withsupercharged zinc Alpha 2-HS glycoprotein (5 μM) resulted in thecondensation and fragmentation of DNA (stained by Hoeschst dye), whichare demonstrated by a more intense fluorescence and breakage of nuclei.As shown in FIG. 14, the nuclei of HT-29 cells incubated with 5 μMsupercharged zinc Alpha 2-HS glycoprotein for 4 hours show a “crescent”shape. The DNA condensation, DNA fragmentation and peripheral crescentsof the nuclei are characteristics of a cell under apoptosis. Apoptosisoccurred in 90-95% of the HT-29 cells. This is in contrast to thecontrol cells which were incubated with saline solution for four hours(stained by Hoeschst dye) in FIG. 15. FIG. 16 further shows the effectsof incubating HT-29 cells with 5 μM supercharged zinc Alpha 2-HSglycoprotein over a period of 4 hours. The HT-29 cells demonstrate bothcell shrinkage and cells round-up which are characteristics ofapoptosis. This, again, is in stark contract to the control cells inFIG. 17.

[0231] As shown in FIG. 18, incubation of 5 μM supercharged zinc Alpha2-HS glycoprotein for 4 hours in LNCaP cells likewise resulted in 90-95%cell death as demonstrated by cell shrinkage and detachment versus thecontrol cells in FIG. 19. In addition, as shown in FIG. 20, incubationof 5 μM supercharged zinc Alpha 2-HS glycoprotein for 4 hours in Hep G2cells resulted in 90-95% cell death as demonstrated by cell shrinkageand cells round-up versus the control cells in FIG. 21.

[0232] 9d. Supercharged Zinc Alpha 2-HS glycoprotein Does Not Affect CCD18Co Cells

[0233] Supercharged zinc Alpha 2-HS glycoprotein has no effect on CCD18Co cells. Incubation of 5 μM supercharged zinc Alpha2 -HS glycoproteinin CCD 18Co cells for six hours did not cause cell death. As shown inFIG. 22, the cells are flat and healthy and could not be differentiatedfrom those of the control in FIG. 23. Note that under the sameconditions and concentrations of supercharged zinc alphs2-HSglycoprotein, apoptosis occurred in 90-95% of LNCaP, HT-29 and Hep G2cells as demonstrated in Section 9c.

[0234] 9e. Zinc Is Necessary For Alpha 2-HS Glycoprotein To InduceApoptosis

[0235] Pre-incubation of Alpha 2-HS glycoprotein with EDTA (withoutrecharging with zinc) completely removed the apoptosis-inducingactivity. HT-29 cells were incubated with 5 μM Alpha 2-HS glycoprotein(without zinc). After four hours, no apoptotic activity was seen. Inaddition, pre-incubation of Alpha 2-HS glycoprotein with EDTA and thenrecharging with calcium did not reveal any apoptosis-inducting activity.HT-29 cells were again incubated with 5 μM calcium charged Alpha 2-HSglycoprotein. After four hours, no apoptotic activity was seen, and thecells appeared normal and healthy. These results further demonstrate theimportance that zinc plays in the selective apoptotic activity ofAlpha2-HS glycoprotein.

[0236] 9f. Supercharged Zinc Alpha 2-HS Glycoprotein Induced ApoptosisIn A Dose Dependent Manner

[0237] Supercharged zinc Alpha 2-HS glycoprotein induced apoptosis in adose dependent manner. As the concentrations increased, so did theapoptotic affect. LD₅₀ at 6 hours were ascertained for HT-29, Hep G2,and LNCaP and are estimated to be 0.75 μM, 0.87 μM and 1.5 μMrespectively.

[0238] 9g. Supercharged Zinc Alpha 2-HS Glycoprotein Fragments alsoSelectively Induce Apoptosis

[0239] When supercharged zinc Alpha 2-HS glycoprotein is dried under avacuum, fragments are generated. (See Section 5a.) The dried fragmentswere reconstituted in 50 μl water. This fragment solution was passedthrough a molecular sieve membrane having a molecular weight cut-off of10,000 Daltons. The resulting filtrate of fragments was collected andtested on cells in an apoptosis assay. This filtrate had an LD₅₀ at 6hours on HT-29 cells of 0.7 μM.

[0240] As demonstrated herein, supercharged zinc Alpha 2-HSglycoprotein, along with active fragments of supercharged zinc Alpha2-HS glycoprotein whether manufactured from modification of naturalAlpha 2-HS glycoprotein, recombinantly, or synthetically, selectivelyinduce apoptosis.

[0241] The scope of the subject invention includes not only the specificnucleotide sequences depicted herein, but all equivalent nucleotidesequences coding for molecules with substantially the same selectiveapoptotic activity.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211>LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Bovine <222> LOCATION:300..309 <223> OTHER INFORMATION: Polypeptide fragment from treatment offetuin from bovine sera as described in the specification. <400>SEQUENCE: 1 His Thr Phe Ser Gly Val Ala Ser Val Glu 1 5 10 <210> SEQ IDNO 2 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Bovine <222>LOCATION: 311..317 <223> OTHER INFORMATION: Polypeptide fragment fromtreatment of fetuin from bovine sera as described in the specification.<400> SEQUENCE: 2 Ser Ala Ser Gly Glu Ala Phe His 1 5 <210> SEQ ID NO 3<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Human <222> LOCATION:300..309 <223> OTHER INFORMATION: Polypeptide fragment from fetuin.<400> SEQUENCE: 3 His Thr Phe Met Gly Val Val Ser Leu Gly 1 5 10 <210>SEQ ID NO 4 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Pig <222>LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptide fragment fromfetuin. <400> SEQUENCE: 4 His Ser Phe Ser Gly Val Ala Ser Val Glu 1 5 10<210> SEQ ID NO 5 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Sheep<222> LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptide fragmentfrom fetuin. <400> SEQUENCE: 5 His Thr Phe Ser Gly Val Ala Ser Val Glu 15 10 <210> SEQ ID NO 6 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Rat <222> LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptidefragment from fetuin. <400> SEQUENCE: 6 His Thr Phe Ser Gly Val Ala SerVal Glu 1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: Mouse <222> LOCATION: 300..309 <223> OTHER INFORMATION:Polypeptide fragment from fetuin. <400> SEQUENCE: 7 His Ala Phe Ser ProVal Ala Ser Val Glu 1 5 10

I claim:
 1. A method of inducing apoptosis in cancer cells by administering Alpha 2-HS glycoprotein to said cancer cells.
 2. The method of claim 1 wherein said Alpha 2-HS glycoprotein has been supercharged with zinc.
 3. A method of inducing apoptosis in cancer cells by administering a peptide fragment of Alpha 2-HS glycoprotein preselected for its apoptotic activity.
 4. A process for preparing supercharged zinc Alpha 2-HS glycoprotein from Alpha 2-HS glycoprotein, which is suitable for treatment on cancer cells comprising: a. incubating Alpha 2-HS glycoprotein in solution with a chelating agent; b. isolating naked Alpha 2-HS glycoprotein from step a. c. incubating the naked Alpha 2-HS glycoprotein in solution with Zinc Acetate; and d. isolating supercharged zinc Alpha 2-HS glycoprotein from the solution in step c. 